Hybrid microSPECT-CT facilitates quantitative analysis of radiotracer-based images of peripheral angiogenesis

Abstract

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Objectives: Previous studies by our group have demonstrated the feasibility of noninvasive imaging of αv integrin to assess temporal and spatial changes in peripheral and myocardial angiogenesis. Despite the substantial progress in molecular imaging technology there is still a relative lack of easy-to-use, reproducible, and accurate software for analysis of microSPECT-CT images. In this study we validate the accuracy and reproducibility of a new image analysis tool (www.bioimagesuite.org) and report the applicability to analyze microSPECT-CT images of peripheral angiogenesis in a widely employed murine model of hindlimb ischemia. Methods: Mice (n=7) underwent surgical occlusion of the right femoral artery to induce unilateral hindlimb ischemia. At 7 days post ligation, 99mTc-labeled radiotracer targeted at αv integrin was injected and 75 min later in vivo microSPECT-CT imaging performed. After imaging mice were euthanized and tissue from distal hindlimb was excised for gamma well counting of radiotracer activity, and ischemic-to-nonischemic (I/N) ratio calculated. MicroSPECT-CT images were analyzed twice (for intraoperator variability) by two independent operators (for interoperator variability). The microCT image was used for segmentation of the microSPECT image. First bisectional planes were placed at each major joint to separate proximal and distal muscle groups, then the regions of bone were excluded from soft tissue using a threshold approach. ROIs derived from microCT image were applied on microSPECT image to calculate mean counts and I/N ratio. Results: Imaging analysis correlated well with the gamma well counting results (r2=0.693). Reproducibility of imaging analysis evaluated by assessment of intraoperator and interoperator variability has shown a very strong positive correlation (r2=0.991 and r2=0.994, respectively). Conclusions: A new semiautomated analysis of microSPECT-CT images provides a noninvasive quantitative tool for evaluation of peripheral angiogenesis. The accuracy and high operator-independent reproducibility of this approach allows for analysis of serial targeted molecular images of lower extremities in animals, which may have implications for clinical imaging in patients with PAD.